Methods for detecting cell apoptosis

ABSTRACT

Methods for the biochemical and immunohistochemical detection of cell apoptosis are described. The methods utilize the detection and measurement of polypeptide fragments generated during apoptosis. Conditions associated with apoptosis may be detected by the methods of this invention. Methods are also presented for the screening of potential therapeutic compounds which inhibit or stimulate apoptosis. Kits for detection of apoptosis and diagnosis of diseases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority under 35 U.S.C. § 119(e) toprovisional patent application Ser. No. 60/030,961, filed Nov. 15, 1996,hereby incorporated in its entirety by reference.

FIELD OF THE INVENTION

The present invention is directed to the detection and quantification ofcell apoptosis. More particularly, the present invention is directed tothe detection of cell apoptosis by using specific antibodies to detectcells undergoing apoptosis and cells which have undergone apoptotic celldeath.

BACKGROUND OF THE INVENTION

Apoptosis is a physiological mechanism of cell death which involves thefragmentation of a cell into membrane-bound particles. The process ofapoptosis is involved in a variety of normal and pathogenic biologicalevents, both during development and in adulthood. Agents which affectapoptosis may have therapeutic utility in treating diseases anddisorders characterized by aberrant cell proliferation or death(reviewed in Hoeppner et al., Biochim. Biophys. Acta 1242: 217-220,1966; Thompson, Science 267:1456-1462, 1995). Techniques for detectionof apoptosis may be useful to screen for potential therapeutic agentsthat may induce or prevent apoptosis.

In view of the biological importance of apoptosis, there exists a needfor methods to specifically detect cells undergoing apoptosis and thosewhich have suffered apoptotic cell death. These methods are crucial tothe identification, characterization, and diagnosis of diseasesdistinguished by abnormal apoptosis, and to the screening of potentialtherapeutic agents that may induce or prevent apoptosis.

Several methods are known for the detection of apoptosis in vitro and invivo, but these have significant drawbacks which limit their utility.Commonly, apoptosis is characterized by condensation and margination ofnuclear chromatin, and fragmentation of nuclear structure into so-calledapoptotic bodies. This apoptotic morphology can be observed usingconventional stains, dyes which selectively accumulate in nuclei such aspropidium iodide or Hoechst 33258, or by electron microscopy (e.g.,Nicoletti et al., J. Immunol. Methods 139:271-279 1991; Crompton et al.,Biochem. Biophys. Res. Commun. 183:532-537 1992; Frey, Cytometry21:265-274 1995; Woo, N. Engl. J. Med. 333:18-25 1995). Unfortunately,these techniques are either of insufficient specificity or are toolaborious and technically complex for the routine selectiveidentification and quantification of apoptotic cells in situ.

Recent attempts to identify and quantify apoptosis have taken advantageof the internucleosomal fragmentation of DNA which is often linked to,but is not diagnostic for, cell death by apoptosis. Various in situhistochemical techniques have been applied to the end-labeling of nickedDNA (Gavrieli et al., J. Cell Biol. 119:493-501 1992; Wijsman et al., J.Histochem. Cytochem. 41:7-12 1993; Wood et al., Neuron 11:621-632 1993).Although these techniques have become popular for marking apoptoticcells in situ, it has become recognized recently that DNA fragmentationcan also result from cell stress or necrotic degeneration. Consequently,the in situ techniques which detect fragmented DNA are not selective indetecting cells undergoing apoptosis (Nitatori et al., J, Neurosci.15:1001-1011 1995; Lassmann et al., Acta Neuropathol, 89:35-41 1995).

Molecular techniques have also been employed for the detection in celland tissue extracts of internucleosomal DNA degradation linked toapoptosis (Wyllie, AH, Nature 284:555-556 (1980); Wyllie et al., J.Pathol. 142:67-77 (1984)). The in situ and molecular techniques whichrely on the detection of internucleosomal DNA fragmentation are notsufficiently thorough for the detection of apoptotic cell death sincethey do not detect forms of apoptosis not associated withinternucleosomal DNA degradation (Cohen et al., Biochem. J. 286:331-3341992; Schulze-Osthoff et al., J. Cell Biol. 127:15-20 1994). Moreover,the molecular methods lack the sensitivity and cellular resolutionneeded to define the role of apoptosis of particular cell types indisease processes. This is especially true for chronic slow degenerativediseases, in which cell death is protracted and asynchronous, andindividual apoptotic cells are present for only a limited period oftime.

An increased understanding of the biochemical mechanisms of apoptoticcell death has arisen from recent genetic and cell biological studies. Afamily of cysteine proteases related to interleukin-lβ converting enzyme(ICE) has been found to play an essential role in the intracellularpathway of apoptosis (reviewed in Martin et al., Cell 82:349-352 1995).ICE itself is not a mediator of apoptosis in most mammalian cell types.Rather, a family of homologous proteases comprising at least nine humanICE family proteases have been identified to date (ICE,CPP32/apopain/Yama, ICH-1, TX/ICH-2/ICE_(rel) III, ICE_(rel) III,MH-1/MH-3/ICE-LAP3, Mch2, FLICE/Mch5, ICE-LAP6/Mch6), each of whichleads to apoptosis when over-expressed in a proteolytically active formin cultured mammalian cells (Miura et al., Cell 75:653-660 1993; Wang etal., Cell 78:739-750 1994; Fernandes-Alnemri et al., J. Biol. Chem.269:30761-30764 1994--Faucheu et al., EMBO J. 14:1914-22 (1995); Kamenset al., J. Biol. Chem, 270:15250-15256 1995, Alnenui et al., J. Biol.Chem. 270:4312-4317 1995; Fernandes-Alnemri et al., Cancer Res.55:6045-6052 1995; Lippke et al., J. Biol. Chem. 271:1825-1828 1996;Muzio et al., Cell 85:817-827 1996; Duan et al., J. Biol. Chem.271:16720-16724 1996). Moreover, treatment of cells with apoptoticstimuli increases ICE-like proteolytic activity in cell extracts (Los etal., Nature 375:81-83 1995; Enari et al., Nature 380:723-726 1996).Proteolytic activity by ICE homologues is required to initiateapoptosis, since overexpression of mutant, inactive ICE homologues doesnot lead to apoptosis, and several protease inhibitors of the ICE familyblock apoptosis (Miura et al., ibid.; Gagliardini et al., Science263:826-828 1994; Enari et al., Nature 375:78-81 1995; Milligan et al,,Neuron 15:385-393 1995, Los et al., ibid.; Zhivotovsky et al., Exp. CellRes. 221:404-412 1995; Schlegel et al., J. Biol. Chem. 271:1841-18441996).

Degradation of specific cellular proteins, as would be expected to occurfollowing the activation of an ICE-like protease, has also beenassociated with apoptosis. For example, poly(ADP-ribose)polymerase(PARP) is cleaved specifically during apoptosis in mammalian cells(Kaufmann et al., Cancer Res, 53:3976-3985 1993) and is an excellentsubstrate in vitro for several ICE homologues (Tewari et al., Cell81:801-809, 1995; Nicholson et al., Nature 376:37-43 1995; Gu et al., J.Biol. Chem. 270:18715-18718, 1995; Fernandes-Alnemri et al., Cancer Res.55:2737-2742 1995-, Fernandes-Alnemri et al., ibid.; Lippke et al., J.Biol. Chem. 271:1825-1828 1996). In the human promyelocytic leukemiacell line HL60 (Collins et al,, Nature 270:347-349 1977), PARP isdegraded in response to incubation with etoposide, which leads to celldeath by apoptosis (Kaufmann et al., ibid). Protease inhibitors whichblock the activity of ICE homologues prevent not only apoptosis, butPARP degradation as well (Schlegel et al., ibid.).

Due to the inadequacies in many of the known methods for the detectionof cell apoptosis, there continues to be a need for new, selectivemethods of detection. The present invention is directed to this, as wellas other, important ends.

SUMMARY OF THE INVENTION

The present invention provides methods for the biochemical andhistochemical detection of apoptosis. The methods are based on the useof antibodies which react selectively with fragments of proteins whosedegradation is stimulated during apoptosis.

In one embodiment, there is provided a method of detecting apoptosis byusing antibodies which bind specifically to peptides or fragments ofproteins having the COOH-terminal amino acid sequence SEQ ID NO: 1(GDEVD) which are generated during apoptosis. The method involvescontacting a sample of cells with the antibody, and determining byimmunoassay the amount of the antibody which binds to the samplerelative to the amount of antibody which binds to a sample known to befree of apoptosis.

Another embodiment provides a method of detecting apoptosis in situbased on the use of immunohistochemical staining of cells in a fixedpreparation. The preparation may be a culture of cells which have beenfixed or a sample of tissue fixed and sectioned. The fixed preparationis contacted with an antibody prepared against the peptide sequence ofSEQ ID NO:2 (CKGDEVD).

In another embodiment of the invention, a first sample of cellscontaining a protein which generates immunoreactive fragments duringapoptosis is exposed to a test compound under conditions or treatmentknown to trigger apoptosis in the cells. A second sample of the samecells is also placed under conditions known to trigger apoptosis, in theabsence of any test compound. A test compound inhibits apoptosis ifantibody binding to the first sample is less than antibody binding tothe second sample.

In still another embodiment, a first sample of cells is exposed to atest compound. A second sample of the same cells is not exposed to thetest compound. A test compound stimulates apoptosis if antibody bindingto the first sample is greater than antibody binding to the secondsample.

In another embodiment, a candidate compound is administered to a mammal,preferably a rodent such as, for example, a gerbil, mouse, or rat, underconditions known to stimulate apoptosis. The level ofapoptosis-generated protein fragments is determined using immunoassaysas described above. A compound tests positive as an inhibitor ofapoptosis if the amount of apoptosis-generated protein fragments takenfrom an animal to which the candidate compound had been administered isless than that present in an equivalent sample from an animal exposed tothe same apoptosis-stimulating conditions, but not exposed to thecandidate compound.

In yet another embodiment, a candidate compound is administered to amammal, such as a gerbil, mouse, or rat, and the level ofapoptosis-generated protein fragments is determined using immunoassaysas described above. A compound tests positive as a stimulator ofapoptosis if the amount of apoptosis-generated protein fragments takenfrom an animal to which the candidate compound had been administered isgreater than that present in an equivalent sample from an animal notexposed to the candidate compound.

Another embodiment of the present invention is the detection in asubject of disease, disorder, or condition associated with cellapoptosis. A sample of cells is obtained from the subject. The sample iscontacted with an antibody against SEQ ID NO:2. The extent of binding ofthe antibody with cells in the sample is determined, preferably byimmunoassay, relative to a control sample known to be free of apoptosis.

Another embodiment of the present invention is an antibody preparedagainst the peptide SEQ ID NO:2 (CKGDEVD). The antibody preferablyreacts selectively with the about 30 kDa NH₂ -terminal polypeptidederivative of poly(ADP-ribose)polymerase produced in cells undergoingapoptosis.

Another embodiment of the present invention is a kit for in vitrodetermination of the level of apoptosis-generated protein fragments in asample. The kit comprises: (1) a primary antibody capable of binding toprotein fragments generated during apoptosis, (2) a secondary antibodyconjugated to a signal-producing label, the secondary antibody being onewhich binds to the primary antibody; alternatively, the secondaryantibody binds specifically to an apoptosis-generated protein fragmentat an epitope different from that to which the primary antibody binds;and (3) a signal-producing tertiary reagent capable of recognizing atagged secondary antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating embodiments of the present invention,there are shown in the drawings certain features. It should beunderstood, however, that this invention is not limited to the preciseembodiments shown.

FIG. 1 shows a design of an antibody which reacts selectively withprotein fragments generated during apoptosis. The sequence around thesite within poly(ADP-ribose)polymerase (PARP) that is cleaved inapoptotic cells is shown at the arrow. The antibodies were designed toreact with the COOH-terminal domain of the NH₂ terminal fragment ofPARP, and other polypeptides carrying the preferred sequence at theircarboxy terminus following cleavage.

FIG. 2 shows an immunoblot detection of apoptotic proteolysis inetoposide-treated HL60 cells. Lane 1--untreated cells; Lane 2--two houretoposide treatment; Lane 3--four hour etoposide treatment. Nuclearextracts prepared from the cells were separated by SDS-PAGE (15 μgprotein per lane) and transferred to nitrocellulose by Western blotting.Polypeptides immunoreactive with Ab127 (1/5,000) were labeled bystandard techniques (e.g., Harlow and Lane (1988) ibid; Roberts-Lewis etal. (1994) ibid). Note that etoposide-treated apoptotic HL60 extracts,but not extracts from untreated cells, contain an immunoreactivepolypeptide of about 30 kDa (arrow), the size expected of the NH₂-terminal fragment of PARP (Lazebnik et al. (1994) ibid.) Levels of animmunoreactive polypeptide of approximately 80 kDa also werereproducibly increased in apoptotic cells, whereas faint immunolabelingof an approximately 100 kDa polypeptide was observed regardless of celltreatment.

FIG. 3 represents confirmation that the about 30 kDa immunoreactivepolypeptide is labeled by Ab127 and is a protein fragment generatedduring apoptosis. Nuclear extracts (10 μg per lane) were prepared frometoposide-treated apoptotic HL60 cells (lanes 1,3) or untreated cells(lanes 2,4). Extracts from cells not treated with the etoposide wereincubated 15 minutes at room temperature with the ICE-like proteaserecombinant human caspase 3 (rhCPP32). Lanes 1,2--immunolabeled withAb127 as described for FIG. 2. Lanes 3,4--immunolabeled with Ab127 thathad been preabsorbed with 1.5 μg peptide immunogen (CKGDEVD--SEQ ID NO:2) per μl antiserum. Note that the immunoreactive about 30 kDapolypeptide in apoptotic cells is also generated by rhCPP32 treatment ofa cell extract not treated with etoposide (compare lanes 1 and 2). Theimmunoreactivity is not observed following preabsorption of theantisense with peptide immunogen (lanes 3, 4).

FIG. 4 shows immunoblot detection of apoptotic proteolysis in growthfactor-deprived neuronally differentiated PC12 cells. NGF-differentiatedPC12 cells were either maintained (+NGF) or withdrawn (-NGF) from growthfactor for 24 hours. Nuclear extracts of differentiated PC12 cells (NUC)were incubated with recombinant CPP32 for either 0, 10, or 40 minutes atroom temperature. For comparison, a nuclear extract of HL60 cells thathad been incubated with CPP32 is also shown. Note that a strongimmunoreactive polypeptide of approximately 46 kDa (and a weak one ofapproximately 35 kDa, not shown in this figure) is detected inNGF-deprived, but not NGF-maintained, PC12 cells. Immunoreactivepolypeptides of identical size are formed in PC12 nuclear extractsincubated with recombinant CPP32. The immunoreactive polypeptides arenot derived from PARP, since they migrate differentially from the about30 kDa PARP fragment apparent in the HL60 extract.

FIGS. 5A and 5B show immunohistochemical detection of apoptoticproteolysis in growth factor-deprived neuronally differentiated PC12cells. PC12 cells either maintained on NGF (5A) or withdrawn from NGFfor 24 hours (5B) were labeled with Ab127 at 1/20,000 using a standardindirect immunoperoxidase technique (e.g., Roberts-Lewis et al. (1994)ibid.) Cells were photographed at 200× magnification, 1260×magnification for the insert. Note that Ab127 strongly labels a subsetof NGF-deprived cells. The immunostaining took the form of a series ofsmall, intensely labeled puncta which sometimes formed an annulus aroundthe cell. No immunolabeling of NGF-deprived PC12 cells was observed inthe absence of Ab127 (data not shown).

FIGS. 6A-6C show immunohistochemical detection of apoptotic proteolysisin the developing post-natal rat brain. Sagittal sections fromaldehyde-fixed rat brain were prepared at 40 μm on a freezing microtome,and were labeled with Ab127 (1/3,000) using a standard indirectimmunoperoxidase technique (Roberts-Lewis et al. (1994) ibid.)A--Hippocampal formation, post-natal day 4 (100× magnification);DG--dentate gyrus; CA3--the CA3 subfield of hippocampus; CA1--the CA1subfield of hippocampus; S--subiculum. Note the scattered, intenselyimmunoreactive cells (open arrowheads). At higher magnification, thesecells have the morphology of neurons. B--Inferior colliculus, post-natalday 9 (200× magnification). Scattered neurons exhibit intenseimmunoreactivity in their perikarya and dendritic processes. Note thebeaded appearance of the immunolabeled dendrites, indicative of activedegeneration. C--Parietal neocortex, post-natal day 9 (100×magnification). Immunoreactivity indicative of apoptotic proteolysis isrestricted to a small subset of neurons in layer 2.

DETAILED DESCRIPTION OF THE INVENTION

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

"Apoptosis" refers to a specific morphological form of cell deathcharacterized by fragmentation of cells and their nucleii intomembrane-bound particles. Apoptosis can be triggered by, for example,treatment with compounds such as etoposide, staurosporine, tumornecrosis factor-α, ceramide, and the like, or by conditions such asx-irradiation.

"About" in reference to a numerical value means ±5% of the indicatedvalue. For example, with specific reference to the NH₂ -terminalpolypeptide derivative of PARP, produced in cells undergoing apoptosis,the molecular weight as determined using Western blot under conditionsbased upon detection with antibodies disclosed herein, is between 30 and31 kDa, i.e., about 30 kDa. (30±5%=a range of between 28.5 and 31.5.)

"Apoptosis-generated protein fragment-specific antibody" refers to anantibody that recognizes apoptosis-generated protein fragments but notintact proteins.

"SEQ ID NO: 1" refers to the amino acid sequence GDEVD(Gly-Asp-Glu-Val-Asp).

"SEQ ID NO:2" refers to the amino acid sequence CKGDEVD(Cys-Lys-Gly-Asp-Glu-Val-Asp).

"Substantially pure" describes a compound which has been separated fromother components which naturally accompany it. Typically, a compound issubstantially pure when at least 75%, more preferably at least 90%, andmost preferably at least 99%, of the total material (by volume, by wetor dry weight, or by mole percent or mole fraction) in a sample is thecompound of interest. Purity can be measured by any appropriate method,e.g., in the case of polypeptides by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. In particular, aprotein is substantially purified when it is separated from the nativecontaminants which accompany it in its natural state.

The terms "reacts selectively" or "binds specifically" describe anantibody that recognizes and binds to target protein fragments, but doesnot substantially recognize and bind to other molecules, such as intactproteins.

"Etoposide" means a semisynthetic derivative of podophyllotoxin, used asan antineoplastic.

"Antineoplastic" means inhibiting the maturation and proliferation ofmalignant cells.

"Epitope" means a site on the surface of an antigen molecule to which asingle antibody molecule binds.

"Vehicle" refers to a relatively inert substance added to a bioactiveagent in order to confer a suitable consistency or form to the agent.

The present invention is directed, in part, to improved methods for thedetection of cell apoptosis. Embodiments of the present inventioninvolve contacting a sample with an antibody which binds selectively toprotein fragments generated during apoptosis. By immunoassay, the amountof the antibody which becomes bound to the target protein fragments isdetermined, using as comparison a sample known to be free of apoptosis.

Poly-ADP-ribose polymerase (PARP) is one of the proteins whoseproteolytic degradation is stimulated in a variety of cells undergoingapoptosis (Kaufmann et al., ibid.; Lazebnik et al., Nature 371:346-3471994; Tewari et al., ibid.). As shown schematically in FIG. 1, inapoptotic cells the approximately 115 kDa PARP is preferentially cleavedat a single site, generating an NH₂ -terminal fragment of about 30 kDaand a COOH-terminal derivative of approximately 85 kDa (apparentmolecular weights as determined by SDS polyacrylamide gelelectrophoresis; Kaufmann et al., ibid; Lazebnik et al., ibid.). Thefive residues immediately NH₂ -terminal to the cleavage site arecompletely conserved in PARP from several vertebrate species, whereasthe residues immediately COOH-terminal to the cleavage site aredivergent (Cherney et al., Proc. Natl. Acad. Sci. USA 84:8370-8374 1987;Saito et al., Gene 90:249-254 1990; Huppi et al., Nuc. Acids Res.17:3387-3401 1989; Ittel et al., Gene 102:157-164 1991; Lazebnik et al.,ibid.).

Additional peptide fragments are produced in cells undergoing apoptosis.For example, DNA replication complex C, DNA-dependent protein kinase,protein kinase of presenilin 1 and 2, spectrin (fodrin), as well asother polypeptides, are known to be degraded and generate peptidefragments during apoptosis. Antibodies to the peptide fragments of thesepolypeptides could be prepared in order to identify cells undergoingapoptosis.

We hypothesized that the highly conserved pentapeptide sequence may becritical for recognition by the protease(s) activated duringapoptosis--i.e., be the apoptosis cleavage recognition site--and anantibody raised to this sequence might react selectively with the about30 kDa NH2-terminal derivative of PARP produced in cells undergoingapoptosis (FIG. 1). Should the sequence be inaccessible in the intactPARP, or in different PARP derivatives formed during the normal turnoverof the protein, then an antibody to the sequence may be expected to be ahighly specific probe for apoptotic proteolysis of PARP. Conceivably,the antibody might also react selectively with fragments of proteinsother than PARP that also contain the particular sequence and,therefore, may exhibit enhanced degradation during apoptosis.

We disclose herein antibodies which preferentially bind to proteinfragments produced during apoptosis and, thus, confirm our hypothesisconcerning the apoptosis cleavage recognition site.

Measurement of apoptosis-associated proteolysis levels can be carriedout by the use of an immunoassay which allows detection of bindingbetween apoptosis-generated protein fragments and an antibody specificto apoptosis-generated protein fragments. The amount of bound antibodycan be determined by detection of enzymic, chromogenic, radioactive,fluorescent or luminescent labels which are attached to either theantibody which binds to the apoptosis-generated protein fragments or toa secondary antibody which recognizes the antibody which binds to theapoptosis-generated protein fragments.

It is preferred that antibodies for use in the methods of the presentinvention react selectively with apoptosis-generated protein fragments,and that they do not react with intact protein. An important feature ofthe present invention is an antibody prepared against the peptideCKGDEVD (SEQ ID NO: 2) that reacts preferentially with the about 30 kDaNH₂ -terminal polypeptide derivative of poly(ADP-ribose)polymerase(PARP) produced in cells undergoing apoptosis. This antibody also reactsselectively with fragments of proteins other than PARP, i.e. theapproximately 46 and approximately 35 kDa polypeptides in apoptotic PC12cells. However, although different proteins may be degraded in apoptoticcells of different sources, thus generating reactive protein fragmentsof different size, if the protein fragments contain the COOH-terminalsequence GDEVD (SEQ ID NO: 1), they will be detected by the methods ofthis invention.

Substantially pure peptides having the amino acid sequences SEQ ID NO: 1(GDEVD) and SEQ ID NO:2 (CKGDEVD) may be used in generating antibodieswhich bind with specificity thereto. Antibodies for the particular aminoacid sequences described herein may be generated by, for example,immunizing a mammal with the appropriate peptide or peptide conjugate.Methods of generating both monoclonal and polyclonal antibodies whichrecognize these peptides are described in Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1988), incorporated herein by reference.

Antibodies which specifically recognize apoptosis-generated proteinfragments, but not intact proteins, can be prepared by standardimmunization methods using a peptide which correspond to a sequencederived from the about 30 kDa NH₂ -terminal fragment of PARP (GDEVD; SEQID NO: 1) as the immunogen. This peptide can be generated by standardmethods known in the art. The NH₂ -terminal extension of SEQ ID NO: 1(GDEVD) utilized to make SEQ ID NO:2 (CKGDEVD) can itself be used as animmunogen. Other protein or peptide antigens which, regardless of theirorigins, length, or degree of homology with naturally occurringapoptosis-generated protein fragments, lead to the production ofantibodies which bind specifically to the apoptosis-generated proteinfragments, may also be used in the methods of the invention. However,preferred antibodies for use in the methods of the present invention areAb127 and Ab128. In order to determine the effectiveness of antibodiesfor use in the methods of the present invention, the reactivities of theantibodies to normally occurring proteins and apoptosis-generatedprotein fragments are compared. Procedures for such comparison areexemplified herein.

The antibodies can be applied to the detection and measurement ofapoptotic proteolysis, to the immunohistochemical tagging andquantification of cells undergoing apoptosis in vitro and in vivo, andto the identification of agents which stimulate or block apoptosis.

The methods of the invention can be used to diagnose cell apoptosis, andassociated conditions, in a subject such as a mammal, and in particulara human subject. The methods can be used to determine whether a subjecthas or is at risk of developing a pathological condition.

The methods of the invention can also be used to diagnose a disease,disorder, or condition which is of either pathological ornon-pathological origin including, but not limited to, chronicneurodegenerative disease, cancer, sepsis, trauma, hypoxia, anoxia,ischemia, spinal trauma, head trauma, lesions, and exposure to toxins.

The method of the present invention is also useful for identification ofcompounds which inhibit or stimulate (e.g., in oncology) apoptotic celldeath by determining that the compounds inhibit or stimulate theformation of apoptosis-generated protein fragments. The proteinfragments are detected by determining the extent of binding of anantibody prepared against the peptide sequence of SEQ ID NO:2 (CKGDEVD).

The methods of the present invention can also be used to determine theextent of activity of a candidate compound in decreasing or increasingthe apoptotic activity in a mammal.

In association with the methods of the present invention, analysis maybe performed on proteins obtained from lysed cells which have beenseparated by means of SDS-polyacrylamide gel electrophoresis ("PAGE").The proteins are contacted with a selective antibody and analysis isperformed, preferably by immunoassay, to determine the presence ofprotein fragments which bind to the antibody. Comparison may be made toa control consisting of proteins from similar lysed cells known to befree of apoptosis.

Immunoassays which may be used in the methods of the present inventioninclude, but are not limited to: ELISA, cell-based ELISA, filter-bindingELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dotblot assays, immunostaining, RIA, scintillation proximity assays,fluorescent immunoassays using antibody conjugates or antigen conjugatesof fluorescent substances such as fluorescein or rhodamine, Ouchterlonydouble diffusion analysis, and immunoassays employing an avidin-biotinor a streptavidin-biotin detection system. Modifications of the knownimmunoassay techniques may also be used.

It will also be understood by one skilled in the art that any of theimmunoassays described above can be used to analyze a tissue sample froma live subject. Possible biological samples for this analysis includeblood cells or biopsied cell or tissue samples which can be obtained bystandard methods. The levels of apoptosis-generated peptide fragments inthe above-described biological samples can be determined in any of theimmunoassays described above employing antibodies that bind specificallyto the peptide sequence GDEVD (SEQ ID NO:1). The level ofapoptosis-generated peptide fragments determined in the biologicalsample from the subject being analyzed is compared to the level found inan unaffected patient, or in a known standard. This method may be usedto diagnose degenerative conditions characterized by abnormal apoptosis.An increase in apoptosis-generated peptide fragments of preferably 50percent, and more preferably 150 percent, compared to control samples,may be considered indicative of a degenerative condition.

The above-described diagnostic assays can be facilitated by the use ofkits which contain the reagents required for carrying out the assays.

The present invention provides kits which may be used in the detectionof cell apoptosis and in the diagnosis of diseases associated therewith.One such kit comprises: (1) a primary antibody capable of binding toprotein fragments generated during apoptosis, (2) a secondary antibodyconjugated to a signal-producing label, the secondary antibody being onewhich binds to the primary antibody; and (3) a signal-producing tertiaryreagent capable of recognizing a tagged secondary antibody.

Another kit that is useful for detection of apoptosis-generated proteinfragments according to the present invention includes (1) a firstantibody capable of binding to protein fragments generated duringapoptosis; and (2) a second antibody conjugated to a signal-producinglabel, the second antibody also being reactive with anapoptosis-generated protein fragment, but one that binds to a sitedifferent from that to which the first antibody binds. This kit is mostsuitable for carrying out a one or two-antibody immunoassay, e.g., asandwich ELISA.

In the assay kits provided according to the present invention, thesignal-producing label linked to the secondary antibody may be, forexample, an enzyme, such as horseradish peroxidase or alkalinephosphatase. Preferably, both the enzyme and the substrate are providedin the kit. The kit may also include an uncoated support onto which asample to be assayed, or the first antibody, can be immobilized.

The present invention is further described in the following examples.These examples are provided for illustrative purposes only, and are notintended to limit the methods and compositions of the present invention.Other suitable modifications and adaptations of the variety ofconditions and parameters normally encountered in immunodiagnostics,apparent to those skilled in the art, are within the scope of thepresent invention.

EXAMPLES Example 1 Preparation Of Antibodies Which React SelectivelyWith Protein Fragments Generated During Apoptosis

Antibodies were prepared by immunizing rabbits with an immunogencomprising the pentapeptide GDEVD (SEQ ID NO: 1) and keyhole limpethemocyanin (KLH). The pentapeptide was directionally conjugated to KLHthrough NH₂ -terminal cysteine-lysine residues (Harlow and Lane (1998)Antibodies, Cold Spring Harbor Laboratory, New York, incorporated hereinby reference), i.e., as the heptapeptide CKGDEVD (SEQ ID NO:2). Theheptapeptide was synthesized by standard solid phase methods, and itsstructure confirmed by fast atom bombardment-mass spectrometry. It wasconjugated to KLH using maleimidobenzoyl-N-hydroxysuccinimide ester(Calbiochem, San Diego, Calif.). Two rabbits were immunized with theimmunogen, and the resultant antisera were collected by standardtechniques. The antisera are referred to as Ab127 and Ab128.

Example 2 Immunoblotting For Detection Of Apoptosis-AssociatedProteolysis In HL60 Cells And Evaluation Of Reactivity Of Ab127 or Ab128With Apoptosis-Associated About 30 kDa PARP Fragment

HL60 cells (obtained from the American Type Culture Collection,Rockville, Md.) were cultured using published techniques (Collins etal., Nature, 270:347-349 (1977), incorporated herein by reference).Cultures of cells were incubated with either 17 micromolar etoposide(from a 1000× stock solution made in dimethylsulfoxide) to triggerapoptosis or a vehicle. After either 2 or 4 hours, cells were lysed inice cold 25 millimolar (mM) HEPES (pH 7.5), 5 mM MgCl₂, 0.1% TritonX-100 and a cocktail of protease inhibitors (1 mM EGTA, 10 microgramsper ml of aprotinin, 10 micrograms per ml of leupeptin, 7 micrograms perml of pepstatin A, 1 mM phenylmethylsulfonylfluoride (PMSF). Theresulting cell lysates were subjected to SDS-polyacrylamide gelelectrophoresis on 4-20% Tris-glycine gradient gels, and the separatedproteins transferred to nitrocellulose membranes by Western blotting.The blots were immunostained with Ab127 (1/5,000) followed by incubationwith alkaline phosphatase conjugated goat anti-rabbit IgG (Bio-Rad,Burlingame, Calif.) and visualized as described previously(Roberts-Lewis et al., J. Neurosci. 14:3934-3944 (1994), incorporatedherein by reference).

As shown in FIG. 2, the antibody labeled three polypeptides in the HL60extracts. Two of the polypeptides were of approximately 100 andapproximately 80 kDa molecular weight, and were labeled irrespective ofcell treatment, although levels of the approximately 80 kDa polypeptideincreased in apoptotic cells. Additionally, Ab127 labeled an about 30kDa polypeptide which was readily detected only in etoposide-treated,but not in vehicle-treated, cells. The level of the about 30 kDaimmunoreactive polypeptide increased with increasing time of cellexposure to etoposide. Stated differently, the antibodies utilizedherein did not bind with the approximately 100 kDa polypeptide or theapproximately 100 kDa polypeptide to the exclusion of the PARPpolypeptide of about 30 kDa.

To control for the specificity of antibody reactivity, preimmune seraand immune sera that were preabsorbed with an excess of the peptideimmunogen were also examined. Preimmune sera from the two rabbits didnot label the about 30 kDa polypeptide. For preabsorption, Ab127 serumwas diluted 50-fold in Tris-buffered saline (pH 7.4), and divided intotwo aliquots. The peptide CKGDEVD was added to one aliquot at 300micrograms/ml, and the two aliquots were incubated at room temperaturefor 1 hour. Next, each aliquot was diluted 100-fold and used forimmunoblotting. As shown in FIG. 3, immunolabeling of the about 30 kDapolypeptide was blocked by Ab 127 preabsorption.

Example 3 Confirmation That The About 30 kDa Immunoreactive PolypeptideIs A Protein Fragment Generated During Apoptosis

Nuclear extracts of HL60 cells were treated with an enzymatically activepreparation of the ICE-like protease CPP32. The baculovirus expressionsystem was used to produce rhCPP32 (Meyer et at., Neurosci. Abstr.22:565 (1996), incorporated herein by reference) and was demonstrated tobe proteolytically active based on cleavage of a synthetic peptidesubstrate (method described in Nicholson et al., Nature 376:37-43(1995), incorporated herein by reference). The protease was purified to˜95% homogeneity by chromatography on a Q-Sepharose FF anion exchangeresin. Nuclear extracts were treated with catalytic amounts of thepartially purified rhCPP32 and incubated at 23° C. for up to 60 minutes.As shown in FIG. 3, incubation with rhCPP32 resulted in an increase inthe level of an Ab127 immunoreactive about 30 kDa polypeptide (lane 2).The labeled polypeptide precisely co-migrated with the immunoreactiveabout 30 kDa polypeptide extracted from intact HL60 cells undergoingapoptosis (lane 1). Preabsorption of the antibody with the peptideCKGDEVD eliminated reactivity with the about 30 kDa polypeptide. Theresults indicate that the apoptosis-associated degradation of PARP canbe specifically detected by an antibody directed at the GDEVD cleavagelocus at the COOH-terminus of the about 30 kDa fragment.

Example 4 Use Of Immunoblotting To Detect Apoptotic Proteolysis InNeuron-Like Cells

The cell line PC12 differentiates into a sympathetic neuron-like cell inresponse to nerve growth factor (NGF), and dies by apoptosis followingNGF deprivation (Batistatou et al. (1993) J. Neurosci. 13, 4433-4438). Aprotease inhibitor of the ICE family has been found to reduce theNGF-deprivation induced apoptosis (Troy et al. (1996) Proc. Natl. Acad.Sci. 93, 5635-5640). Accordingly, reactivity of Ab127 and Ab128 withpolypeptides was compared in NGF-maintained PC12 cells and NGF-deprivedapoptotic PC12 cells.

PC12 cells were maintained in NGF-containing medium under low serumconditions (0.18% fetal bovine serum) for 7 days. NGF withdrawal wasinitiated by three washes with NGF-free culture medium, followed byaddition of a neutralizing antibody to NGF (Boehringer Mannheim,Indianapolis, Ind.). After various time intervals following NGFdeprivation, apoptotic cell death was evaluated by measurements of cellsurvival, internucleosomal DNA fragmentation, and chromatincondensation. Immunoblotting with Ab127 (FIG. 4) or Ab128 (data notshown) was performed as described in Example 2 above, and demonstratedthat, by 8 hours following NGF deprivation, two immunoreactivepolypeptides of approximately 46 and approximately 35 kDa appeared inthe cells deprived of NGF (FIG. 4). The content of these polypeptidesincreased with longer periods of NGF deprivation.

Example 5 Confirmation That The Approximately 46 And Approximately 35kDa Immunoreactive Polypeptides Are Protein Fragments Generated DuringApoptosis

Cell-free nuclear extracts of PC12 were incubated with recombinant humanCPP32, the ICE-like protease as described in Example 3 for HL60 cellextracts. Incubation of nuclear extracts of PC12 cells with CPP32 led toa time-dependent increase in the content of approximately 46 andapproximately 35 kDa polypeptides which co-migrated with twoimmunoreactive species obtained from NGF-differentiated PC12 cellsdeprived of NGF (FIG. 4). Migration of the two polypeptides isdistinguishable from the immunoreactive about 30 kDa PARP fragment inapoptotic HL60 cells. The results indicate that the approximately 46 andapproximately 35 kDa polypeptides represent fragments from proteinsother than PARP that contain the GDEVD domain at their COOH-terminus andare specifically generated in PC12 cells undergoing apoptosis. It willbe recognized by one skilled in the art that Ab127 or Ab128 may be usedfor affinity purification of these ICE-like protease substrates andtheir identification by protein microsequencing or mass spectrometrymethods.

Example 6 Immunohistochemical Detection Of Cell Death By Apoptosis

NGF-differentiated PC12 cells were grown on chambered microscope slideswhich had been pre-treated with poly-ornithine and laminin. After 7days, the culture medium was changed and half of the cultures weredeprived of NGF as described in Example 4. After 24 hours, cells wereprocessed for immunohistochemistry using Ab127 as follows. Cells werefixed in 2% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4(60 minutes), and incubated sequentially in 0.09% H₂ O₂ in phosphatebuffer (20 minutes), followed by 10% normal goat serum/0.5% TritonX-100/Tris-buffered saline (TBS) for 60 minutes. Cultures were incubatedin TBS alone or TBS containing Ab127 at dilutions from 1/10,000 to1/40,000 for 2 hours. Following 3 washes in TBS, all cultures wereincubated in biotinylated anti-rabbit IgG (Vector, Burlingame, Calif.)diluted 1/200 in TBS. After 30 minutes, the cultures were washed in TBSand incubated with the avidin-biotin-peroxidase complex (ABC Elite kit,Vector) according to the manufacturer's instructions. Following severalwashes in TBS, color development was carried out using a standard nickelintensified diaminobenzidine method. After rinsing, cultures of cellswere placed under cover slips and photomicrographs were taken using aNikon Microphot system.

As shown in FIG. 5, only very light background staining was observed forAbb 127-stained PC12 cells maintained on NGF (FIG. 5A). Thus, Ab127 didnot react with proteins in healthy PC12 cells that were maintained onNGF. NGF-deprived cells labeled in the absence of the Ab127 primaryantibody showed no immunoreactivity. In NGF-deprived cultures, manycells did not label with Ab127 above background, but a subset of cellswas intensely immunolabeled (FIG. 5B). This specific staining was onlyobserved with Ab127 treatment, and took the form of clusters of darklystained puncta, which often appeared to form a ring (FIG. 5B, insert).The pattern of immunolabeling is similar to the appearance of apoptoticbodies forming from the disintegration of the plasma and nuclearmembranes in cells undergoing apoptosis.

Similar results were obtained with other cells. Hippocampal neuronsgrown in primary culture were stimulated to die by application of theneurotoxic metabolic poison 3-nitropropionic acid. Independent labelingof the cultures with the nuclear stain Hoechst 33258 revealed thepresence of apoptotic neurons, with condensed chromatin fragmenting intoapoptotic bodies, in the midst of viable cells. When the cultures wereimmunostained with Ab127, most of the cells were not labeled, but asubset of neurons was intensely immunoreactive. In contrast, no Ab127immunoreactive cells were found in control cultures that had not beentreated with the neurotoxin.

Example 7 Immunohistochemical Labeling Of Apoptosis-AssociatedProteolysis In Developing Rat Brain

To examine the use of Ab127 as a probe for identifying cells undergoingapoptosis in situ, Ab127 immunohistochemistry was performed on neonatalrats. It has been shown that excess numbers of neurons are formed duringearly rat brain development, and significant programmed neuronal deathoccurs by apoptosis during the first 2 post-natal weeks (reviewed inOppenheim (1991) Annu. Rev. Neurosci. 14, 453-501). To examine theutility of Ab127 in identifying cells undergoing apoptosis in situ,Ab127 immunohistochemistry was performed on neonatal rats.

Brains of rats 1, 4, 7 and 9 days of age were fixed in 4%paraformaldehyde, cryoprotected, sectioned in the sagittal plane at 40micrometers on a sliding microtome, and stained for Ab127immunoreactivity using a standard indirect immunoperoxidase procedure(described in, e.g., Roberts-Lewis et al., J. Neurosci. 14:3934-3944(1994), incorporated herein by reference). The Ab127 was used atdilutions ranging from 1/1,000 to 1/50,000. Control sections wereprocessed as described in Roberts-Lewis et a., above, except that theprimary antibody (Ab127) was omitted. Following immunostaining, sectionswere mounted onto glass slides, dehydrated, delipidated, placed undercover slips, and examined and photographed using a Nikon Microphotsystem.

The immunostaining with Ab127 of the developing rat brain revealed ahighly restricted distribution of intensely immunoreactive cells. Nearlyall brain cells during the first 10 post-natal days were devoid of Ab127immunoreactivity. However, groups of cells within specific brain regionsand at particular developmental stages were heavily immunopositive.

FIG. 6 shows the immunolabeling of subsets of cells in the parietalcortex, hippocampal formation, and inferior colliculus. As shown in FIG.6A, the post-natal day 4 hippocampus contains intensely immunoreactivecells scattered among several subfields. FIG. 6B illustrates the heavyimmunostaining of neurons of the inferior colliculus at post-natal day9. The neurons are in an acute stage of degeneration, as manifested bythe beaded, fragmented appearance of their dendrites. As shown in FIG.6C, only rare neocortical cells were immunoreactive. In parietal cortex,scattered neurons at an acute stage of degeneration were labeled, almostexclusively in layer 2. At a later stage of development, fewerimmunopositive neurons were detected in the neocortex, hippocampus, andother brain regions, consistent with these neurons having the shortresidence time characteristic for cells dying by apoptosis. However, bypost-natal day 9, numerous intense immunopositive cells appeared in thecorpus callosum. Their location and morphology identify theimmunolabeled cells as oligodendroglia.

No immunolabeling was observed in either neurons or oligodendrocytes insections stained in the absence of Ab127. The results confirm and extendthe observations reported above for cultured cells, and indicate that anantibody directed at the preferred ICE-homolog proteolytic cleavage siteGDEVD may be used to selectively label cells undergoing developmentalapoptosis in situ.

All references cited herein are hereby incorporated by reference intheir entireties.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Gly Asp Glu Val Asp                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Cys Lys Gly Asp Glu Val Asp                                              1               5                                                            __________________________________________________________________________

What is claimed is:
 1. A method of detecting apoptosis in cellscomprising the steps of: (a) contacting a sample of cells with anantibody which binds specifically to protein fragments generated duringapoptosis, wherein said antibody binds specifically to an epitope onsaid protein fragments comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1 and SEQ ID NO:2; (b) determining byimmunoassay the amount of said antibody which binds to said sample; and(c) comparing the amount of antibody bound in step (b) with the amountof said antibody which binds to a sample known to be free of apoptosis,wherein if said amount in step (b) is greater than said amount in saidsample known to be free of apoptosis, then apoptosis is detected.
 2. Amethod of detecting apoptosis in cells or tissue in situ comprising thesteps of: (a) contacting a fixed preparation of said cells or tissuewith an antibody which binds specifically to protein fragments generatedduring apoptosis, wherein said antibody binds specifically to an epitopeon said protein fragments comprising an amino acid sequence selectedfrom group consisting of SEQ ID NO: 1 and SEQ ID NO:2; (b) determiningby immunohistochemical analysis the amount of said antibody which bindsto said preparation; and (c) comparing the amount of antibody bound instep (b) with the amount of said antibody bound to a fixed preparationof cells or tissue not undergoing apoptosis, wherein if said amount instep (b) is greater than said amount in said cells or tissue notundergoing apoptosis, then apoptosis is detected.
 3. The method of claim1 or 2 wherein said protein fragments are produced from poly-ADP-ribosepolymerase.
 4. The method of claim 3 wherein said protein fragments areNH₂ -terminal fragments of about 30 kDa.